Structures and roles of the polymorphic forms of tobacco mosaic virus protein: VI. Assembly of the nucleoprotein rods of tobacco mosaic virus from the protein disks and RNA

The assembly of tobacco mosaic virus involves a preformed protein aggregate, the disk, which consists of two rings each of 17 protein subunits, as the sole protein source. The kinetics of this assembly have been studied, using both tobacco mosaic virus RNA, which causes a rapid initiation and so enables growth to be studied, and also polyadenylic acid, with which initiation is slowed down and thus can be partially resolved from growth. Two disks interact with a special nucleotide sequence at the 5′-hydroxyl end of a single tobacco mosaic virus RNA molecule to initiate the formation of the viral nucleoprotein helix, which then grows by the addition of further disks. All of the subunits from these further disks are incorporated into the helix, so that growth proceeds by the co-operative addition of 34 subunits at a time. Under the conditions used, rearrangement of each disk takes about six seconds, giving a total time for the growth of a complete virus particle of just over six minutes.     

Structure and function of disk aggregates of the coat protein of tobacco mosaic virus

Experiments have been carried out on the coat protein of tobacco mosaic virus (TMVP) to test for the occurrence of the previously postulated RNA-induced direct switching, during in vitro assembly of tobacco mosaic virus (TMV), of the subunit packing from the cylindrical bilayer disk to the virus helical arrangement. No evidence was found for such RNA-induced switching and no evidence for the direct participation of the bilayer disk in either the nucleation or elongation phases of the in vitro virus assembly. Instead, virus assembly proceeds by an initiation step involving the binding of the RNA to the previously characterized two-plus turn helical aggregate that is formed from small oligomers of subunits. However, a bilayer disk, which has been characterized in high ionic strength crystals, has been observed in low ionic strength virus assembly solutions only as a transient species upon depolymerization of dimers of bilayer disks formed in solution at high ionic strength, and not as an equilibrium species of TMVP.     

Structures and roles of the polymorphic forms of tobacco mosaic virus protein. VII. Lengths of the growing rods during assembly into nucleoprotein with the viral RNA

The length distributions of growing particles have been determined and followed as a function of time during the reconstitution of tobacco mosaic virus from its isolated RNA and protein. The protein was supplied either largely as the “disk” aggregate or as A-protein obtained by cooling a disk preparation. In a further experiment, the growth was initiated with disks and then continued with A-protein. It has been possible to correct the resulting distributions of lengths for the effect of broken RNA molecules and hence to obtain a picture of the distribution of lengths of the growing particles.From these distributions and also the average lengths, it is concluded that the growth is most rapid when disks are the protein source, giving full length particles in six to ten minutes. When A-protein is supplied for the growth, the rate is about one quarter of that with disks, irrespective of whether the rods have been nucleated with disks or not.     

Circular dichroism and sedimentation studies on the reconstitution of tobacco mosaic virus

Reconstitution of tobacco mosaic virus from its constituents, the coat protein and RNA, was investigated by means of ultracentrifugation and circular dichroism measurement. Tobacco mosaic virus protein forms a 20S double-layer disc under conditions favorable for tobacco mosaic virus reconstitution. Dibromination of the tyrosine 139 residue of tobacco mosaic virus protein prevents formation of the 20S disc.Acidification of the tobacco mosaic virus protein solution causes 20S discs to polymerize into long helical rods. Changes in the CD spectra of tobacco mosaic virus protein in the near-ultraviolet region suggest that stacking of the aromatic sidechains of amino acid residues stabilizes the helical rod. The dibrominated tobacco mosaic virus protein also has the ability of rod elongation under acidic condition. CD studies reveal that assembly of tobacco mosaic virus particles from its constituents is stabilized by the stacking effect between the base residues of RNA and the aromatic residues of tobacco mosaic virus protein.Cucumber green mottle mosaic virus protein, which acts as a substituent for tobacco mosaic virus protein in tobacco mosaic virus reconstitution, was also investigated.     

RNA-protein interactions in the assembly of tobacco mosaic virus.

Assembly of tobacco mosaic virus is initiated by the binding of a specific loop of the RNA into the central hole of the disk aggregate of protein subunits. Since the nucleation loop is located about five-sixths along the RNA molecule, subsequent elongation must be bidirectional. We have now measured the rates of elongation in the two directions by determining the lengths of RNA protected from nuclease digestion at different times and using either intact TMV rNA, or RNA with most of the longer tail removed. Comparison of the rates with the protein supplied as either a mixture of disks with A-protein (a mixture of less aggregated states) or just A-protein, shows that different mechanisms and protein aggregates are used for the most rapid growth. When disks are present, they add more rapidly along the longer RNA tail but do not appear to add directly on the shorter tail. In contrast, smaller aggregates (A-protein) can add at both ends of the rod, but do so more slowly. Mechanisms for these processes are discussed. Preliminary results on the binding of the specific hexanucleotide AAGAAG to the disk are given and compared with the known changes on binding nonspecific hexanucleotides or the trinucleotide AAG.     

Protein Synthesis Directed by the RNA from a Plant Virus in a Normal Animal Cell

RNA from tobacco mosaic virus can be translated inside oocytes of the frog Xenopus laevis. The main product is a polypeptide with a molecular weight of 140,000. There is no evidence for coat protein synthesis, and it is unlikely that the polypeptide that is made contains either a whole or a partial coat protein sequence.The picture of translation of tobacco mosaic virus RNA obtained using oocytes is very much simpler than that found using cell-free protein-synthesizing systems, in which a great many polypeptides are made under the direction of tobacco mosaic virus RNA. The reasons for this difference are discussed, and the relative merits of in vivo and in vitro protein-synthesizing systems are compared.     

Oligonucleotide binding to the coat protein disk of tobacco mosaic virus. Possible steps in the mechanism of assembly

Binding of the oligoribonucleotides AAG, AAGAAG and AAGAAGUUG to the disk aggregate of tobacco mosaic virus coat protein has been studied in solution under conditions favourable for virus assembly. The two longer oligomers bind strongly with Kd around 1 microM, approach complete saturation of binding sites and cause the formation of long, nicked helical rods resembling the virus. It is suggested that the binding of these oligomers, with sequences chosen from the assembly origin of the viral RNA, simulates the tobacco mosaic virus assembly process. No binding could be detected for AAG, indicating that chain length is a crucial determinant in the interaction. The binding of AAGAAG to coat protein crystals is very much weaker than that observed in solution, and the crystals crack at high oligomer concentrations. The corresponding oligodeoxyribonucleotide, d(AAGAAG), shows no binding to the protein in solution; the interaction is extremely specific for RNA.     

Location and encapsidation of the coat protein cistron of tobacco mosaic virus. A bidirectional elongation of the nucleoprotein rod.

The coat protein cistron of tobacco mosaic virus has been located on the viral RNA starting between 975 and 1050 nucleotides from the 3'-hydroxyl end. This locates its 5' end close to the origin for virus assembly, where the first protein disk interacts with RNA. It also means that the coat protein mRNA must have a short 5'-untranslated tail and a long (over 500 nucleotides) 3' one. The recovery of characteristic oligonucleotides in nuclease-protected rods during the growth from RNA and a protein disk preparation shows that elongation of the nucleated rods proceeds independently in both directions though, on average, much more rapidly along the longer 5' tail than the shorter 3' tail. Protected RNA of length equal to that in the complete virion is first seen within 6 min, showing that the most rapidly elongated particles are substantially complete by this time.     

Direct visualization of the RNA location in cucumber green mottle mosaic virus and tobacco mosaic virus protein disk

The location of RNA in cucumber green mottle mosaic virus and tobacco mosaic virus protein disks was visualized by a negative staining method as a narrow ring localized at a radius of 4 nm, which corresponds to the location of RNA obtained by X-ray diffraction studies of tobacco mosaic virus. The same ring-shaped stains were observed in the end views of helical rods prepared in acidic solutions from viral protein without RNA. Since such a ring-shaped image could not be observed in end views of natural particles and reconstituted particles composed of protein and RNA, the narrow ring was concluded to indicate the RNA location on the basis of X-ray analysis.     

Cucumber mosaic virus coat protein-mediated protection

Transgenic tobacco plants (CP +) that express the coat protein gene of cucumber mosaic virus (CMV)-Y strain were highly protected from infection with either CMV virions or CMV RNA, while transgenic protoplasts were also protected from infection with CMV virions but not with CMV RNA. CP + plants showed greater susceptibility to infection with satellite RNA-free CMV-Y than CMV-Y containing satellite RNA. At temperatures above 30°C, CP + plants did not or poorly resist infection with CMV. Elevated temperature affected the accumulation of CP rather than its mRNA, suggesting that CP molecules are mainly involved in virus resistance in CP + plants.     

Structural comparisons of the aggregates of tobacco mosaic virus protein

The coat protein of tobacco mosaic virus forms numerous aggregates, including the small A-protein, the disk, and two helical forms. The structures of the disk, the helical protein forms, and the virus are compared. Most of the differences are in the conformation of the chain between residues 89 and 113, which lies in the region of protein at the center of the virus, inside the RNA. It is disordered in the disk, but has a fixed conformation in the virus and the protein helices. The differences between the virus and the two helical protein forms are largely in the conformations of arginines and carboxylic acids in this region.     

Self-assembly of tobacco mosaic virus: the role of an intermediate aggregate in generating both specificity and speed

The tobacco mosaic virus (TMV) particle was the first macromolecular structure to be shown to self-assemble in vitro, allowing detailed studies of the mechanism. Nucleation of TMV self-assembly is by the binding of a specific stem-loop of the single-stranded viral RNA into the central hole of a two-ring sub-assembly of the coat protein, known as the 'disk'. Binding of the loop onto its specific binding site, between the two rings of the disk, leads to melting of the stem so more RNA is available to bind. The interaction of the RNA with the protein subunits in the disk cause this to dislocate into a proto-helix, rearranging the protein subunits in such a way that the axial gap between the rings at inner radii closes, entrapping the RNA. Assembly starts at an internal site on TMV RNA, about 1 kb from its 3'-terminus, and the elongation in the two directions is different. Elongation of the nucleated rods towards the 5'-terminus occurs on a 'travelling loop' of the RNA and, predominantly, still uses the disk sub-assembly of protein subunits, consequently incorporating approximately 100 further nucleotides as each disk is added, while elongation towards the 3'-terminus uses smaller protein aggregates and does not show this 'quantized' incorporation.     

Poliovirus RNA-Dependent RNA Polymerase Synthesizes Full-Length Copies of Poliovirion RNA, Cellular mRNA, and Several Plant Virus RNAs In Vitro

The poliovirus RNA-dependent RNA polymerase was active on synthetic homopolymeric RNA templates as well as on every natural RNA tested. The polymerase copied polyadenylate. oligouridylate [oligo(U)], polycytidylate . oligoinosinate, and polyinosinate. oligocytidylate templates to about the same extent. The observed activity on polyuridylate. oligoadenylate was about fourfold less. Full-length copies of both poliovirion RNA and a wide variety of other polyadenylated RNAs were synthesized by the polymerase in the presence of oligo(U). Polymerase elongation rates on poliovirion RNA and a heterologous RNA (squash mosaic virus RNA) were about the same. Changes in the Mg(2+) concentration affected the elongation rates on both RNAs to the same extent. With two non-polyadenylated RNAs (tobacco mosaic virus RNA and brome mosaic virus RNA3), the results were different. The purified polymerase synthesized a subgenomic-sized product RNA on brome mosaic virus RNA3 in the presence of oligo(U). This product RNA appeared to initiate on oligo(U) hybridized to an internal oligoadenylate sequence in brome mosaic virus RNA3. No oligo(U)-primed product was synthesized on tobacco mosaic virus RNA. When partially purified polymerase was used in place of the completely purified enzyme, some oligo(U)-independent activity was observed on the brome mosaic virus and tobacco mosaic virus RNAs. The size of the product RNA from these reactions suggested that at least some of the product RNA was full-sized and covalently linked to the template RNA. Thus, the polymerase was found to copy many different types of RNA and to make full-length copies of the RNAs tested.     

Protein-RNA interactions during TMV assembly

A review of the structural studies of tobacco mosaic virus (TMV) is given. TMV is essentially a flat helical microcrystal with 16 1/3 subunits per turn. A single strand of RNA runs along the helix and is deeply embedded in the protein. The virus particles form oriented gels from which high-resolution X-ray fiber diffraction data can be obtained. This may be interpreted by the use of six heavy-atom derivatives to give an electron density map at 0.4 nm resolution from which the RNA configuration and the form of the inner part of the protein subunit may be determined. In addition, the protein subunits form a stable 17-fold two-layered disk which is involved in virus assembly and which crystallizes. By the use of noncrystallographic symmetry and a single heavy-atom derivative, it has been possible to solve the structure of the double disk to 0.28 nm resolution. In this structure one sees that an important structural role is played by four alpha-helices, one of which (the LR helix) appears to form the main binding site for the RNA. The main components of the binding site appear to be hydrophobic interactions with the bases, hydrogen bonds between aspartate groups and the sugars, and arginine salt bridges to the phosphate groups. The binding site is between two turns of the virus helix or between the turns of the double disk. In the disk, the region proximal to the RNA binding site is in a random coil until the RNA binds, whereupon the 24 residues involved build a well-defined structure, thereby encapsulating the RNA.     

Disk aggregates of tobacco mosaic virus protein in solution: electron microscopy observations

Previous studies of the coat protein of tobacco mosaic virus (TMVP) have shown that TMVP presumably exists as linear stacks of two-ring cylindrical disks in the 0.7 M ionic strength buffer used for crystallizing the disks for X-ray diffraction studies [Raghavendra, K., Adams, M.L., & Schuster, T.M. (1985) Biochemistry 24, 3298-3304]. The spectroscopic and sedimentation studies of solutions of TMVP under these crystallizing conditions have demonstrated a long-term metastability of these disk aggregates when they are placed in 0.1 M ionic strength buffers, as are used for reconstituting tobacco mosaic virus from TMVP and viral RNA. The present work describes an electron microscopic study of TMVP disk aggregates under the same solution conditions employed in the previous spectroscopic and sedimentation studies. The results show that in the pH 8.0 0.7 M ionic strength crystallization buffer TMVP exists as stacks of disks which range in size from about 6 to 24 layers, corresponding to 3-12 2-layer disk aggregates having 17 subunits per layer. These TMVP aggregates persist in a metastable form in 0.1 M ionic strength virus reconstitution buffer with no apparent changes in structure of the stacked disks. The results are consistent with the conclusions of the solution physical-chemical studies which suggest that the disk structure may not be related to the 20S TMVP aggregate that is the nucleation species in virus     

Leadzyme formed in vivo interferes with tobacco mosaic virus infection in Nicotiana tabacum

We developed a new method for inhibiting tobacco mosaic virus infection in tobacco plants based on specific RNA hydrolysis induced by a leadzyme. We identified a leadzyme substrate target sequence in genomic tobacco mosaic virus RNA and designed a 16-mer oligoribonucleotide capable of forming a specific leadzyme motif with a five-nucleotide catalytic loop. The synthetic 16-mer RNA was applied with nontoxic, catalytic amount of lead to infected tobacco leaves. We observed inhibition of tobacco mosaic virus infection in tobacco leaves in vivo due to specific tobacco mosaic virus RNA cleavage effected by leadzyme. A significant reduction in tobacco mosaic virus accumulation was observed even when the leadzyme was applied up to 2 h after inoculation of leaves with tobacco mosaic virus. This process, called leadzyme interference, is determined by specific recognition and cleavage of the target site by the RNA catalytic strand in the presence of Pb(2+).     

The isolation of tobacco mosaic virus RNA fragments containing the origin for viral assembly

Upon mixing purified TMV RNA with limited amounts of viral coat protein in the form of the disk aggregate, a unique region of the whole RNA becomes protected from nuclease digestion. The protected RNA consists of fragments up to 500 nucleotides long in varying yields, which are found in nucleoprotein particles having a protein-nucleic acid ratio similar to the mature virus. The protected RNA, when reextracted, is able to rebind to coat protein disks rapidly, quantitatively and with high affinity, becoming once more RNAase-resistant in the process. Small aggregates of TMV protein (A protein) are inactive in formation of the nuclease-resistant complexes. On the basis of this evidence, we identify the isolated RNA fragments as portions of TMV RNA containing the origin or initiation site for in vitro reassembly, which have been protected from digestion by incorporation into assembly nucleation complexes.The yield, but not the length distribution, of the protected RNA pieces is found to double upon increasing the protein added from 1–2 disk-equivalents of protein per RNA molecule. This implies that the formation of the nucleation complexes may involve a highly cooperative initial addition of protein.